US5919625AExpiredUtility

Ribonuclease resistant viral RNA standards

82
Assignee: AMBION INCPriority: Jul 3, 1996Filed: Apr 29, 1997Granted: Jul 6, 1999
Est. expiryJul 3, 2016(expired)· nominal 20-yr term from priority
Inventors:Dwight DuboisMatthew WinklerBrittan Pasloske
C12Q 1/707C12N 15/88C12N 2795/10142C07K 14/005C12Q 1/706C12N 2740/16022C12P 19/34C12N 15/86C12N 2795/10122C12Q 1/702C12N 15/10C12Q 1/6851C12N 7/00
82
PatentIndex Score
72
Cited by
188
References
41
Claims

Abstract

The present invention is directed to the process of creating a recombinant nucleic acid standard which is resistant to ribonuclease digestion and is non-infectious. A single strand of recombinant nucleic acid is encapsidated by bacteriophage proteins. The recombinant nucleic acid is a hybrid sequence encoding bacteriophage proteins and a specific non-bacteriophage sequence. A non-bacteriophage RNA sequence can be used as an RNA standard to help quantify the number of RNA molecules in an unknown sample. The recombinant RNA in its packaged form is highly resistant to ribonucleases, insuring that the RNA standard is not compromised by inadvertent ribonuclease contamination. These ARMORED RNA™ standards are ideal as RNA standards for the quantification of RNA viruses such as HIV and HCV from human body fluids such as blood and cerebrospinal fluid.

Claims

exact text as granted — not AI-modified
What is claimed is: 
     
       1. A method of assaying for the presence of an RNA of interest in a sample, comprising: 1) obtaining a sample to be assayed;   2) obtaining a ribonuclease resistant RNA standard comprising, encapsidated in a bacteriophage viral coat protein, an RNA segment comprising a segment encoding a sequence that serves as a standard in detection or quantification of the RNA of interest;   3) admixing the sample with the RNA standard;   4) isolating RNA from the admixture; and   5) assaying for the presence of the RNA with a method comprising testing for the presence of the segment encoding a sequence that serves as a standard in detection or quantification of the RNA of interest as a quantitative or qualitative standard in determining the presence or quantity of the RNA of interest.   
     
     
       2. The method of claim 1, wherein the viral coat protein is a modified viral Coat Protein. 
     
     
       3. The method of claim 1, wherein the bacteriophage viral, coat protein is of an E. coli bacteriophage of genetic subclass A. 
     
     
       4. The method of claim 1, wherein the bacteriophage viral coat protein is of an E. coli bacteriophage in serological group I. 
     
     
       5. The method of claim 1, wherein the bacteriophage viral coat protein is of an MS2/R17 bacteriophage. 
     
     
       6. The method of claim 1, comprising testing for the presence of the sequence that serves as a standard in detection or quantification of the RNA of interest as a quantitative standard in determining the quantity of the RNA of interest in the sample. 
     
     
       7. The method of claim 1, comprising testing for the presence of the sequence that serves as a standard in detection or quantification of the RNA of interest as a qualitative standard in determining the presence of the RNA of interest in the sample. 
     
     
       8. The method of claim 1, wherein the ribonuclease resistant RNA standard is a bacteriophage. 
     
     
       9. The method of claim 1, comprising testing for the presence of the sequence that serves as a standard in detection or quantification of the RNA of interest as a positive control. 
     
     
       10. The method of claim 1, comprising testing for the presence of the sequence that serves as a standard in detection or quantification of the RNA of interest as an internal standard. 
     
     
       11. The method of claim 1, comprising testing for the presence of the sequence that serves as a standard in detection or quantification of the RNA of interest to determine the concentration of the RNA of interest. 
     
     
       12. The method of claim 1, further comprising a viral Maturase protein. 
     
     
       13. The method of claim 12, wherein the viral Maturase protein is a modified viral Maturase protein. 
     
     
       14. The method of claim 13, wherein the recombinant RNA segment comprises an Operator coding sequence and a viral Maturase protein binding site. 
     
     
       15. The method of claim 14, wherein the Operator coding sequence is a modified Operator coding sequence. 
     
     
       16. The method of claim 14, wherein the recombinant RNA segment further comprises a viral Maturase protein coding sequence. 
     
     
       17. The method of claim 14, wherein the recombinant RNA segment further comprises a viral coat protein coding sequence. 
     
     
       18. The method of claim 17, wherein the viral Coat Protein coding sequence codes for a bacteriophage viral coat protein. 
     
     
       19. The method of claim 18, wherein the bacteriophage viral Coat Protein is a modified bacteriophage viral coat protein. 
     
     
       20. The method of claim 1, wherein the segment encoding a sequence that serves as a standard in detection or quantification of the RNA of interest is a sequence that serves in detection or quantification of an RNA of diagnostic value. 
     
     
       21. The method of claim 20, wherein the segment encoding a sequence that serves as a standard in detection or quantification of the RNA of interest serves as a standard in detection or quantification of HIV-1, HIV-2, HCV, HTLV-1, HTLV-2, hepatitis G, an enterovirus, or a blood-borne pathogen. 
     
     
       22. The method of claim 20, wherein the segment encoding a sequence that serves as a standard in detection or quantification of the RNA of interest serves as a standard in detection or quantification of HIV-1, HIV-2, or HCV. 
     
     
       23. The method of claim 20, wherein the segment encoding a sequence that serves as a standard in detection or quantification of the RNA of interest serves as a standard in detection or quantification of HIV-1. 
     
     
       24. The method of claim 23, wherein the segment encoding a sequence that serves as a standard in detection or quantification of the RNA of interest serves as a standard in detection or quantification of HIV-1 and comprises a modified HIV-1 sequence. 
     
     
       25. The method of claim 1, wherein the ribonuclease resistant RNA standard comprises a recombinant RNA segment comprising a segment encoding a sequence that serves as a standard in detection or quantification of the RNA of interest. 
     
     
       26. The method of claim 25, wherein the recombinant RNA segment is resistant to ribonuclease. 
     
     
       27. The method of claim 25, wherein the recombinant RNA segment comprises at least two viral Maturase binding sites. 
     
     
       28. The method of claim 25, wherein the recombinant RNA segment comprises at least two Operator sequences. 
     
     
       29. The method of claim 25, wherein the recombinant RNA segment comprises an Operator sequence, a sequence coding a viral Maturase protein, and a non-bacteriophage sequence. 
     
     
       30. The method of claim 25, wherein the recombinant RNA segment comprises an Operator sequence, a sequence coding a viral Maturase protein, a sequence coding a viral coat protein and a non-bacteriophage sequence. 
     
     
       31. The method of claim 25, wherein the recombinant RNA segment comprises an Operator sequence, at least two viral Maturase binding sites, a sequence coding a viral Maturase protein, a sequence coding a viral coat protein and a non-bacteriophage sequence. 
     
     
       32. The method of claim 25, wherein the recombinant RNA segment comprises an Operator sequence, at least two viral Maturase binding sites, a sequence coding a viral Maturase protein, a sequence coding a viral coat protein, a non-bacteriophage sequence, and a sequence coding a Replicase protein. 
     
     
       33. The method of claim 25, wherein the recombinant RNA segment comprises a bacteriophage sequence from an RNA bacteriophage and a segment encoding a non-bacteriophage sequence that serves as a standard in detection or quantification of the RNA of interest. 
     
     
       34. The method of claim 33, wherein the non-bacteriophage sequence that serves as a standard in detection or quantification of the RNA of interest is a non-bacteriophage viral sequence. 
     
     
       35. The method of claim 33, wherein the bacteriophage sequence is a sequence from an E. coli bacteriophage of genetic subclass A. 
     
     
       36. The method of claim 33, wherein the bacteriophage sequence is a sequence from an E. coli bacteriophage in serological-group I. 
     
     
       37. The method of claim 33, wherein the bacteriophage sequence is a sequence from an MS2/R17 bacteriophage. 
     
     
       38. The method of claim 25, wherein the recombinant RNA segment comprises a sequence coding a Replicase protein. 
     
     
       39. The method of claim 38, wherein the sequence coding the Replicase protein codes a modified Replicase protein that is not active. 
     
     
       40. The method of claim 25, wherein the recombinant RNA segment comprises an Operator sequence and a viral coat protein sequence. 
     
     
       41. The method of claim 40, wherein the recombinant RNA segment further comprises a non-bacteriophage sequence.

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